胚胎电转protocol

PROTOCOLInvivoelectroporationintheembryonicmousecentralnervoussystemTetsuichiroSaitoDepartmentofDevelopmentalBiology,GraduateSchoolofMedicineChibaUniversity,Chiba260-8670,pondenceshouldbeaddressedtoT.S.(tesaito@)Publishedonline9November2006;correctedonline7and29December2006(detailsonline);doi:10.1038/nprot.2006.276©2006

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/natureprotocolsThisprotocoldescribesabasicryofelectricpulsesfollowingmicroinjectionofDNAintothebrainventricleorthespinalcordcentralcanalenableseffiectionisfacilitatedbyforceps-typeelectrodes,whichholdtheuterusand/an90%ofoperatedembryossurviveandmorethan90%pressioninneuronspersistsforalongtime,evenatpostnatalstages,,thismethodcouldbeusedtoanalyzerolesofgenesnotonlyinembryonicdevelopmentbutalsoinhigherorderfunctionofthenervoussystem,UCTIONAnalyzinggenefunctioninmiceThestudyofgenefunctionandnetworksactivityinthebraininvivoisakeyissue,andmanynovelgeneshavebeenidentifienicandgenetargetingtechniqueshavegeneratednumerousmoinantviruses,liposomes2andr,transfectionbyliposomesorbiolisticsismostlylimitedtotissuesadjacenttothevascularsystemandsuperfiuctionoftransgenicandgener,itisnotalwayseasytoexpressageneatthetimeandplacerequired,asthenumberoftranscriptionalregulatorysequences,suchasenhancersandsilencers,thatarea,quickandeasymethodsofgenetransferinmicewillgelectroporation:anoverviewElectroporationhasbeenwidelyusedtointroduceDNAintovarioustypesofcellsandtissues:prokaryotic4andeukaryoticcells5,6,mousemuscle7,inovochickembryos8,cases,electricpulsesaredeliveredinasolutioncontainingcells,usly,wehaveshownthatgenescanbesuccessfullytransfectedevenintocellsthatarenotintheproximityofelectrodesbydeliveringelectricpulsestothemouseembryofromoutsidetheuterus,andthatthetrvivoelectroporationhasbeensuccessfullyappliedtoanalyzegenefunc-tion10–16andtranscriptionalregulation14,method,DNAismicroinjectedintothebrainventricleorthespinalcordcentralcanaloftheembryo,anddembryossurviveintheuterus,retransfectedintocellsthatarelocatedadjacenttotheventricleorthecentralcanal10,11,ofthetransfectedcellsareneuronalprogenitors,theyandtheirdescendantneurons1552|VOL.1NO.3|2006|heembryomoreclearlyduringtheprocedure,edembryosthatareconnectedtotheplacentacansurviveintheyolksacinsidethepregnantmouse,oporationoutsidetheuterinewallisdescribedasexouteroelectroporation,whereasinuteroelavebeensuccessfullytransfectedtothetelencephalon10,13,15–19,dien-cephalon10,midbrain10,hindbrain20andspinalcord11,12,agesofinvivoelectroporationInvivoelectroporationhasthefollowingadvantageousfeatures:electroporationectionefficiencyishigh(seeTable1).However,despitethishighefficiency,ificantincreaseincelldeathhasbeendetectedafterelectroporation(ref.18;TS,unpublishedresults).Multiplegenesondifferentplasmidscanbesimultaneouslytransfectedintothesamecells10,inrast,transferofmultiplegenesisnoteasyforrecombinantviralsystems,becadsthatarelargerthan10kbcanbesuccessfullytrans-fected10,esideoftheventriclethatisclosesttotheanodeistransfected10,atureisusefulforanalyzinggenefunction,asionofatransfectedgeneislimitedtoparticulartypesofneuerebralcortex,progenitorsthataretransfectedataparticularstagegiverisetoneuronsofspecificlayers10,ctgenescanbeexpressedbyneuronsofdifferentlayersbydoubleelectroporationattwodifferentstages18.

PROTOCOLTABLE1|nicstageE11.5E12.5E13.5E15.5Voltage(V)22304045Survivingembryos(%)70.8±6.486.9±5.396.1±1.492.2±7.8EYFP+embryos(%)82.6±2.397.2±2.893.8±2.597.8±ineeficiencyofinvivoelectroporation,2mlof0.5mgmlÀ1ofareporterplasmid,pCAG-EYFP,alandEYFP+rates(percentages)werecalculatedforeverylitterfromthenumberofsurvivingembryos/operatedandEYFP-positive/survivingembryos,aarerepresentedasmean±,enhancedyellowfledinvivoembryossurviveformonths,incontrastwithinvitroembryos,pressioninpostmitoticneuronsusuallypersistsforalongtime,uptoabout4monthsafterelectroporation,possisistentgeneexpressioninneuronsimplicatesthatthismethodcaeptioniscerebellargranulecells,whichcontinuetodivideactivelyevenatpostnatalstages,andexpressionlevelsoftransfectedgenesdecreaseinaccordancewiththeirdivisions(DKawauchiandTS,unpublishedresults).Purifitouspromoters,suchastheCAGpromoter,logyandmigrationofneuralprogenitorsandtheirdescendantneuronsareclearlyvisualizedbyusingafluorescentproteingenedownstreamofthepromoter,aghtransfectioncanbeconfinedtoareasclosetotheinjectionsiteofDNA10,geneexpressioncanbefurlectro-poration,thenestinandSox2enhancersactivategeneexpressionspecificallyinneuralprogenitorcells17,1enhancerfunctionsinMath1-expressingcells14,analysisoftranscriptionalregulation,whichmostlyrequiredconstructionoftransgenicmice,rmore,quantitativeassaysoftranssaysareveryuseful,ygainoffunctionhasbeenrepressedinchicks©2006

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/natureprotocolsly,mammaliangeneshavebeenknockeddownbyusinginvivoelectroporationtotransfectcellswithshortinterferingRNAs16andplasmidsthatproduceshorthairpinRNAs22,sionofatransfectedgenehasbeenrestrictedtoaspecificembryonicstagebyrecombination-basedeliminationofthegeneusingtheCrerecombinase–rly,genescouldbespatio-temporallyknockedoutbyelectroporatingCreintomicecarryingthegenesthatarefltisandretinaaretransfectedafterDNAinjectionintotheseminiferoustubule24andsubretinalspace25,djacenttoaclosedspacethatdoesnotallowextensivedilutionoftheinjectedDNA(suchastheventricle)willbegoodtargetsforinvivoelectroporation,tionsofinvivoelectroporationAdrawbackofinvivoelectroporationisthatstrongelectricpulsesaffectheartrhythm,arstobe-ring-typeelectrodes,whichweredesignednottocovertheheart,rdrawbackofthetechniqueisthatgeneexpressionislimitedtotheoperatedmiceandisnottransferredtotheiroffspring,,weprovideadetailedprotocolforcarryingoconstruct(lessthan14kbinsizesofartested)canbetransfectedusingthismethod,,invivoelectroporationisaquickandrelativelysimpletechniquethatcanbereadilymodifi(seeREAGENTSETUP)..EndoFreePlasmidKit(Qiagen,.12362,12381and12391).TE:10mMTris-HCl(pH7.5),1mMEDTA.70%ethanol/30%TE(vol/vol).3Msodiumacetate(NaOAc).Ethanol.1mMTris-HCl(pH7.5),0.1mMEDTA.10ÂPBS:1.37MNaCl,27mMKCl,100mMNa2HPO4,20mMKH2PO4.10%Nembutal(Nembutal(AbbottLaboratories)/saline(1:9,vol/vol)).!bestoredinalockedlocationanditsusemustbeproperlyrecorded..70%ethanol(ethanol/H2O(7:3,vol/vol)).Saline,carmine(DaiichiPharmaceutical)apillarymicrohematocrittube(DrummondScientific,75mm).Micropipettepuller(SutterInstrument,.P-97/IVF).Watchmaker’s#ingboard:an85mmÂ135mmplasticboardwithfoursmallholes(distancesbetweenholesareshowninFig.1),forceps(NatsumeSeisakusyo,.A14).Ringforceps(NatsumeSeisakusyo,.A26).Peristalticpumpandtube:anytypeofpumpandtubecanbeused,iftheyarefitwithwarmsalinedelivery(seebelow)EQUIPMENTNATUREPROTOCOLS|VOL.1NO.3|2006|1553

PROTOCOLREAGENTSETUPMousestrainsICRmice(Clea,Japan)areusedinmostcases,becausetheybearmany,usuallymorethanten,ousestrains,suchasC57BL/6,nofadaywhenavaginalplugisfoundisdesignatedasembryonicday(E)ofbirthisdesignatedpostnatalday(P)0.!CAUTIONAllexperimentsshouldbeperformedinaccordancewithationofplasmidDNAPlasmidsarepurifiedusingtheEndoFreePlasmidKitaccordingtothemanufacturer’sprotocolwiththefollowingminormodifications:(i)washtheQIAGEN-tipcapturingDNAwithBufferQC(washbufferinthekit)threetimes,insteadoftwice;(ii)after70%ethanolrinse,suspendtheDNApelletwithasmallamount(300mlfortheMaxiKit)ofTEandprecipitateDNAagainwith1/fuge,air-dryandsuspendtheDNApelletwith1mMTris-HCl(pH7.5)invivoelectroporation,dilutetheDNAsolutionwith10ÂPBSandH2Otoafinalconcentrationof30–300nM(0.1–1mgmlÀ1,ifpCAG-EYFPisused)-EYFPcarriestheEYFP(enhancedyellowfluorescentprotein)genedownstreamoftheCAGpromoter,CALPlasmids,suchaspCAG-EYFP,thatcarryafluorescentproteingenewillbeusefultoevaluatetransfectionefficiency,becausetheirfllevelsofgeneexpressionareobtainedbyinjectionofhigherconcentrationsofDNA,andexpressionlevelsappeartoplateauat150nM(0.5mgmlÀ1,ifpCAG-EYFPisused).PurifiedplaENTSETUPPreparationofmicropipettesforDNAinjectionPullglasscapillarymicro-hematocrittubesusingthemicropipettepuller,P-97/IVF,underthefollowingconditions:pressure,500;heat,800;pull,30;velocity,40;time,,breakpulledpipettestoanB60mmexternaldiameterbypinchingthemwithwatch-maker’s#ipsofthepipetteswithawater-resistantmagicmarkerinordertofinesevery5mmonthepipettesusingthemarkertomeasurethevolumeofinjectedsolution—linedeliveryWarmsalineisdroppedfromtheendofatube(Fig.2a)byaperistalticpump(Fig.2b).Theotherendofthetubeisinsertedintoasaline-containingbottle(Fig.2c)ina371Cbathincubator(Fig.2d).pshouldbecontrolledbyanon/offfootswitchtomakeyourhandsfree.75

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/natureprotocolsFigure1|bisfixedbypullingdowntherubberbandthroughthehole..Bathincubator:-controlledpipette(DrummondScientific).oporator:CUY21Edit(Nepagene)orElectroSquarePoratorT820(BTX).Switchbox(Nepagene,902).Forceps-typeelectrodes(Nepagene,650P3,dsuture(NatsumeSeisakusyo,.F17-50braidedsilk).therdetails,see/class/dev/protocol/650P10,diametersofwhichare3,5and10mm,respectively)salinedelivery(seebelow)PROCEDUREPreparationofanimalsforinvivoelectroporation1|Anesthetizeatimed-pregnantmousewithanintraperitonealinjectionof10%Nembutalsolution.2|helimbsthroughtherubberbandsandfixinpositionbypullingrubberbandsdownwards(Fig.1).Placeastackofpapertowelsundertheoperatingboardtoabsorbspilledsaline.3|Covertheabdomenwithapieceoffoldedgauze(B70mmÂB150mm)thathasanB30-mm-longslitinitscenter.4|Drenchthegauzewith70%ethanol.5|PinchtheskinthroughtheslitwithcurvedforcepsandmakeanB30-mm-longmidlineincisionthroughtheskinandthentheabdominalwallwithscissors.6|Electroporationcanbecarriedoutinutero(optionA)orexoutero(optionB).mCRlsobeusedforembryosolderthanE12.5,visedtomasterinuteroelectroporationfirst,becauseexouteroelectroporationrequiresmorecarefulhandlingoftheembryo.(A)Inuteroelectroporation(i)Attachringforcepstotheuterusatagapbetweenembryosandcarefullypulltheuterusoutoftheabdom-inalcavity(Fig.3).Becarefulnottoattachtheforcepstotheplacentaorembryos.1554|VOL.1NO.3|2006|NATUREPROTOCOLSacbdFigure2|Warmsalinedelivery.

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/natureprotocolsFigure3|4|CALSTEPDuringsurgery,totheplacentaandbloodvesselsofthemesometriumshouldbeminimized;ESHOOTING(ii)Inject1–2mlDNAsolutionintothebrainventricleorthespinalcordcentralcanalusingthemouth-controlledmicropipetteundertheilluminationofafiberopticlightsource(Fig.4).mCRITICALSTEPItisstronglyadvisedtomasterinjectionfi,suchasIndigocarmine,isusefulforpractice,becauseitenablesthesiteandportanttoknowhowandwheretoinsertthemicropipettebasedonthelocationsofembryonicbrainbloodvessels,14.5embryosfirst,hardtofindinjectionsites,practicshouldbeusedonlyforpractice,ESHOOTING(iii)HoldtheDNA-injectedembryothroughtheuterus,paralleltotheembryonicanteroposterioraxis,withforceps-typeelectrodes(Fig.5)anddeliverthreetofictricpulsesaredeliveredeverysecond,lvoltagesareshowninTable1.!CAUTIONDonotuseextreandafterholdingtheembryo,theelectrodesddamagetotheplacentaandbloodvesselsofthemesometrium,urfaceoftheuterusisdrybeforedeliveringelectricpulses,betransfectedintobothsidesoftheventricle,ifnecessary,bydeliveringthreeorfourCALSTEPChooseappropriateelectrodesthatcoveraregionwheregenetransferisdesired,ESHOOTING(iv)CALSTEPTheuterusshouldbepeeledgentlyandcarefullyfromthegauzebydrenchingitwithwarmsaline,ESHOOTING(v)Filltheabdominalcavitywithwarmsaline.(vi)CALSTEPTheabdominalwallshouldbesuturedclosedsufficientlytightlytopreventleakageofsaline.(vii)Warmthemouseinametalcageonaslidewarmerat381C,5|TheinuteroembryoisPROTOCOLS|VOL.1NO.3|2006|1555

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/natureprotocolsFigure6|7|CALSTEPThebottomofthemetalcagemusttightlyattachtothetopoftheslidewarmerforefficientheattransmission.(viii)Transferthemousetoastandardcage.(B)Exouteroelectroporation(i)Pulluptheuteruswithwatchmaker’s#CALESHOOTING(ii)Inject1–2mlDNAsolutionintothebrainventricleorthespinalcordcentralcanal(Fig.6).mCRITICALSTEPIfasmallamountofliquidinsidetheyolksacleaksthroughaholemadebyinjection,r,ESHOOTING(iii)HoldtheDNA-injectedembryothroughtheyolksac,paralleltotheembryonicanteroposterioraxis,withforceps-typeelectrodesanddeliverelectricpulsesasinStep6A(iii)(Fig.7).TROUBLESHOOTING(iv)RepositiontheembryointotheabdominalcavitycarefullyasinStep6A(iv).ESHOOTING(v)Filltheabdominalcavitywithwarmsaline,closetheabdominalwallandskin,andwarmthemouseasinSteps6A(v)–(viii).mCRITICALSTEPIfyouwanttoexaminegeneexpres-sionatpostnatalstages,pupsmustberecoveredbycesariansectionatE19.5andrearedbyafostermother.7|CALSTEPForaninitialevaluationoftransfectionefficiency,analyzeembryonicsurvivalandexpressionofacontrolgene,suchasEYFP,2daysafterelectroporation,ESHOOTINGTIMINGWeusuallyoperateonallembryosandcompletethesurgery(fromincisiontoclosureoftheskin)gery